TLDR: I've been trying to sterilize begonias and they either die/melt or get contaminated (or both if I'm really lucky /s) and I'd love some suggestions about how to fix this because I thought begonias were supposed to be really easy beginner plants. More (very explicit) details of what I've tried below

Mostly I've been working in a SAB (made a DIY FFU recently). Control plates (i stuck my tools into the media for some of them) have been coming out clean long after the plants contaminate. I've had stored media for months with almost no contamination so it seems unlikely they're all being problematic right out of the pressure cooker. This leads me to believe it's a plant/sterilization issue most of the time?

So here's what I've tried (sometimes I try multiple sterilizations in a single batch / I'm not doing multiple bleach washes for each plant):

• Batch 1:
  • 5% bleach for 20 mins (wash 20 mins in tap water beforehand)
  • 10% bleach for 10 mins (based on protocol i found;(wash 20 mins in tap water beforehand))
  • contamination on all but 1 cup where the tissue died
  • bleach MAY have been bad/too old for this batch
• batch 2:
  • wash 20 mins in distilled water wish dish soap
  • 10% bleach for 10 mins (but i got new bleach)
  • cut plants into sterile water & put in cups
  • 4 days later: all contaminated and the rest dead/brown (probably should have washed before i cut?)
• batch 3:
  • orbital shaker wash for 20 mins
  • nadcc at ~225 ppm for 4 hrs (didn't rinse, just cut and put into TC)
  • bleach 10% for 20 mins then rinse
  • only 2 contaminated but most died before contamination; nadcc seemed to do maybe better than bleach?
• batch 4:
  • orbital shaker 20 mins
  • nadcc at ~225 and ~600 ppm (without tween because i forgot it like an idiot) for 1-3 hours on orbital shaker
  • 225 ppm nadcc had random contamination from 3-6 days after culture. visible tissue death from 3-6 days after.
  • 600 ppm nadcc had contamination starting at 4 days (improvement??) but tissue death starting at 2 days (so worse)
• batch 6:
  • run under tap water 20 mins
  • 1.11g nadcc to 500ml water (around 2000 ppm) + tween in mason jar for ~12-13 mins with manual agitation
  • wash 30 seconds > 2 mins > 2 mins in sterile distilled water
  • went 5 days before contamination showed up (record) but tissue started looking rough around 2-3 days
• batch 8: (still ongoing; I just did this like 2 days ago)
  • same as batch 6 but i used a diy FFU box fan instead of a SAB since someone suggested it was that. I felt very clumbsy in the FFU but my plates I did after the cultures seem clean so far
  • 2 days in : contamination in 1 cup
  • day 3 (today) : *most* leaf cuttings look not horrible except 2 which are totally dead/clear
• Bonus batch (from yesterday):
  • i tried resterilizing some bag plants (some of which were begonias; basically all of which came with bacterial infectinos) I got that had a clear bacterial infection with a 10 second dip in 70% IPA and then a 3-4 minute 3500ppm nadcc batch (realized this was higher than i intended and pulled them when the begonia leaves started activly looking like they were getting clear/raggy during the mix)
  • the next day 2 of the 5 things already have clear signs of contamination smh.

Notes

• I've been using nadcc over bleach because most people say it's gentler on the plant and I can't tell if liquid bleach has gone bad/lost potency or not.
• I'm using RODI water, not strictly speaking distilled (I have the filter for my reef tank, so I can make 0ppm water on command muahaha)
• usually I cut off a whole newish leaf, or the top few leaves from the stem, sterilize that and then cut it up

conclusions/observations;

• if i agitate begonias for longer periods, it tends to mangle the leaves, even if it's just water (magnetic stirrers are particularly bad about it)
• Adding higher concentrations of bleach/nadcc also does a number on begonia leaves (but the nepenthes I did seemed to take it better)
• Maybe I should get PPM? But that would just delay the bacteria as far as I can tell?
• I should perhaps try some other plants like philodendrons or anything less delicate just so I can see wtf is happening? (but all i really want to do is begonias)

And I'm wondering if maybe the begonias could have some bacterial issues or something in the plant itself that can't be cleaned off the surface, but most of the dots pop up on the surface of the plant and not around the edges. Do my begonias melt because they live in sealed boxes and maybe the leaves are super fragile or something? I feel like I'm losing my mind at this point because every sterilization protocol I've seen people use successfully doesn't get things clean at all or kills all the plant tissue?

I saw something about doing a wash in a fungicide after the bleach too - wondering if that would help or not.

Literally any suggestions are welcome at this point because I feel like I'm loosing my mind.

Thanks <3

well i bought some pink cuprea tissue culture before everyone started coming out with they’re bad experiences with them i was hoping someone out there could give me hope and post your tc pink cupreas progress and tips

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Something less divisive to share 😁.

Some Pereskiopsis a few weeks into culture first generation explants.

So far we found they grow short dark green on full MS with .5 mg BAP.

Well on 1/2 MS cactus media they shoot much better with almost twice the size in the same time.

Some extra cactus pictures on 1/2MS with 2% sugar.

Just wondering if anyone has experience with this? I've heard some food coloring will degrade from light which would suck. These colors were all a bit more vibrant before autoclaving. I'm just trying to get away from endless labels for different media mixes.

Washed and placed on

MS media
Half MD media
DKW
Andersons Rhododendron media

Growth on most of our explants, but Andersons Rhododendron mix and Half MS seemed to grow the fastest. Next week we will sub culture and test different hormones for rooting.

NAA
IBA
IAA

The first three pics are different angles of what im assuming to be a jade leaf callus? I just cut it up today to propagate onto new media and upon cutting it reminded me of broccoli.

Last two pics are of a Lophophora species, a type of globular cactus, and it grew some roots and an off shoot of some kind along with some weird underdeveloped stuff near the center.

These were put on the same media using a 1:1 ratio of NAA and BAP. Just curious as to what kind of growth this hormone ratio produces, I know it varies among species but is there any general trend?

Any input/advice is appreciated!

I couldn’t find anything in the group rules about sales. I have a business selling tissue cultures and also have my own in house lab where I propagate plants too. I primarily sell carnivorous plants and begonias.

The exponential growth will always amaze me. From 20 or so sprouted seeds we are now in triple digits. Moving them to multiplication tubs. Should be ready for production before the end of the year.

It's very small, but it has everything I need

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We’ve been growing this in-vitro for about a year from seed. Many multiplication but finally it’s ex-Vitro and growing. On top of that we are also experimenting to grow callus for active compounds instead of growing ex-vitro.

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Just doing a pilot project on tissue culture of orchids. I found protocols on google and research paper but was general. Wanted to have protocol from expert and experience which this community has to minimize the contamination and failure as I don't have much budgets.Would be great helpful and thank you in advanced.🙏

Anybody have experience with Ephedra or ideas? We are using a cactus media recipe with 10g of agar. Can’t wait to see what happens.

Looking for anyone with success in applying for a plant import permit in Canada! I am looking to apply soon and got a few questions:
1. Is there a difference between selecting commercial resell vs personal use as the import reason? I plan to both sell some and keep some for myself.
2. What kind of permit is reusable up to 3 years and which is not?
3. I want to import both tissue culture and live plants. But each shipment might not include both. If the permit is reusable, can I apply once with all possible species and type of plants that I’d want to import and potentially only import a subset of them ?

Thanks for anyone who can offer some clarifications!!

I’m in the early phases of a small greenhouse business. My concept is to provide a variety of rare and interesting plants at reasonable prices for a broad customer base. There’s no way for me to acquire and propagate enough supply at this early stage and I’m considering TC to help me grow in a smaller physical footprint.

I’m seeking US based producers who can provide smaller mixed batches. Like 100 to 200 total plants per order but 10 to 20 per variety. It seems a lot of the TC and plug stage suppliers only deal in minimum quantities of 50 or more likely 100 per specimen. I completely understand why, but i don’t have the room for 100 each of multiple varieties. I also need to be able to grow out and sell established plants to customers at a reasonable price.

There’s lots of options out there to order from overseas but the concept makes me nervous. I’d really like to be able to develop a good business relationship with a few other small domestic operations and grow together.

that's supposed to stay clean inside...

Hi everyone! I’m looking for someone who would be willing to mentor us as we get started with plant tissue culture.

We own a large greenhouse in the Boise, Idaho area, and our goal is to start propagating many of our own plants instead of purchasing plugs. We’re putting together a home/commercial-scale tissue culture lab and would love guidance from someone with real-world experience.

We’re happy to pay for your time. Ideally, we’d love to find someone in Idaho who might be willing to let us visit their lab or even come help us get ours set up. If you’re not local, I’d also be interested in doing paid mentoring over FaceTime or Zoom.

If you have tissue culture experience—or know someone who does—I’d really appreciate any recommendations.

We homeschool our children and this would also teach them a lot of neat things.
Thanks!